The naturally occurring cyclic peptide didemnin B displays antineoplastic activity against human tumor cell lines in culture and weak antitumor activity in human patients. Didemnin B was the first marine natural product to enter clinical trials for treating cancer. Unfortunately, it has not fulfilled its early promise in this regard, possibly due to rapid metabolism of the compound in vivo. However, didemnin B also possesses immunosuppressive activity and prolongs graft survival in rats and mice. This immunosuppressive activity occurs at concentrations of didemnin B that are significantly lower than necessary to inhibit cell growth and may be linked to the ability of this compound to halt cell cycle progression at the G1-S border. Structure-function studies of synthetic and naturally occurring didemnins have identified features of these molecules that are important for its different biological properties; these data suggest that the immunomodulatory effects and cytotoxic effects of didemnins may occur through two distinct mechanisms. TITLE: Synthesis and Use of Tritiated Didemnins (Continued) The cytotoxic properties of didemnin B have been attributed to the ability of this molecule to inhibit protein biosynthesis. Consistent with this hypothesis, we have shown that didemnin B inhibits translation in vitro by interfering with ribosomal translocation. Inhibition of translation in vitro requires a protein called elongation factor-1a (eEF-1a), implicating this translation factor as a target for didemnin B binding. In support of this hypothesis, eEF-1a was isolated from brain tissue using a didemnin affinity column, and a didemnin derivative was shown to bind to this protein in a GTP-dependent manner with Kd = 15 mM. Despite these successes, some uncertainty exists regarding the relevance of these findings to the mechanism of action of didemnin B in cells. For example, both the dissociation constant for didemnin binding to eEF-1a, and the IC50 for inhibition of protein synthesis in vitro, are three orders of magnitude higher than the concentration of didemnins required to inhibit cell growth. Moreover, some of the biological effects of didemnins are achieved in the absence of any significant effect upon total protein synthesis; notably (1) didemnin B inhibits T-cell blastogenesis at concentrations approximately three orders of magnitude lower than are required to inhibit protein biosynthesis in the same cells, and (2) vasopressin stimulated myo-inositol uptake by WRK1 (rat mammary tumor) cells is inhibited by nordidemnin at concentrations that have no significant effect upon protein synthesis. These observations may reflect the operation of even more than two mechanisms for didemnins in cells, which would be consistent with the idea that the cytotoxic and immunosuppressive effects of didemnins arise through different pathways. One or more of these pathways must account also for the recent discovery that didemnin B induces programmed cell death in both human and murine tumor cell lines. A second didemnin binding protein has been isolated using an affinity column similar to the one used to isolate eEF-1a. This protein displays protein pamitoyl thioesterase activity and has been linked to the severe brain disorder, infantile neuronal ceroid lipofuscinosis. However, the significance of this protein to the biological properties of didemnins is presently unclear. Thus, despite the clinical potential of didemnins as antitumor and immonusuppressive agents, many questions remain to be answered regarding their mechanism of action. Experiments to address these questions would be facilitated by the availability of radioactive, biologically functional didemnin analogs. The basic molecule was labelled by heterogeneous tritiation of a double bond in the dipeptide fragment, and coupling to the main part of the molecule. The product was then purified by HPLC, and analyzed by proton and tritium NMR spectroscopy.